Pre-mRNA splicing, an essential processes in eukaryotic gene expression, is catalyzed by the spliceosome, a complex ribonucloprotein (RNP) machine composed of five small nuclear RNAs (snRNAs) and numerous proteins. The spliceosome requires an intricate network of RNA-RNA and RNA-protein interactions to correctly align the pre-mRNA substrate for catalysis. This network is established through an elaborate assembly pathway prior to the catalytic step. The complexity of this pathway and the dynamic nature of the spliceosome have obscured our understanding of the splicing mechanism and, in particular, our ability to define the RNA and/or protein components that participate in catalysis. In the proposed research plan, I describe a systematic approach to elucidating the RNA and protein components required for spliceosome catalysis. By selectively removing RNA and protein components from assembled spliceosomes and assaying for splicing defects, effects due to assembly versus catalysis can be uncoupled to identify essential and non-essential RNA and protein components at the catalytic step. Additionally, crosslinking at the catalytic core will identify key protein residues that interact with reactive groups. Based on thes results, I propose reconstituting a minimal, stable spliceosome poised for catalysis. Such a system will be pivotal for future mechanistic and structural analyses.